| Coronary artery disease is one of the primary diseases to endanger human health. Most patients with CAD can be effectually cured by convention medicines or percutaneous transluminal coronary angioplasty(PTCA) or coronary aorta bypass graft(CABG). However, the restenosis rate of PICA is as high as 30%-50% and the long-time results of CABG are not satisfactory at present. On the other hand ,a few of patients with CAD are not suitable to receive the therapy of PICA or CABG, and the myocardial ischemia relief is a problem awaiting solution. How to prevent restenosis and how to promote neovascularization in ischemic myocardium are a new subject in the near future. The object of the current study is to investigate an important growth factor, vascular endothelial growth factor(VEGF), whether to prevent restenosis of percutaneous transluminal angioplaty(PTA) and induce neovascularization in ischemic myocadium. So that to find a new method in the management of coronary heart disease using Gene therapy .This study was divided into three parts. The main methods and results are summary as follow.1. The construction, transcription and expression of eukaryotic expression vector pcDNA3/ hVEGF165. This part was designed to construct an eukaryotic expression vector pcDNA.,/ hVEGF1B5 with biological activity for further gene therapeutic experiment. The hVEGF16ScDNA fragment was cloned into pcDNA., plasmid. The eukaryotic expression vector pcDNA:!/ hVEGF165 was transfected into CHO-K1 cells by liposome mediator. The positive clone was screened using G-418 resistance. The hVEGF165 gene transcription and protein expression were examined by Northern blot and Western blot . The activity of recombinant hVEGF,65 was detemined by the 3H~TdR incorporation assay in mouse vascular endothelial cells in vitro. In result, thespecific bands were found in Northern blot and Western blot in experiment group. In conclusion, the recombinant hVEGF,fiS can be produced from CHO-K1 cells that had been transfected by pcDNA,.,/ hVEGFlfi5 and has biological activity to stimulate vascular endothelial cell growth.2. The effects of pcDNA3/ hVEGFI65 on reendothelialization and restenosis of carotid artery injury induced by angioplaty. A rabbit model of injured carotid artery was established using percutaneous transluminal Angioplasty(PTA). The pcDNA:,/ hVEGFlfiS (SOOug, n=8) and pcDNA3 (SOOug,n=8) were separately transfected into injured arterial wall with 30min incubation . The carotid artery was imaged by aortic angiography at the end of 2-week and 4-week posttreatment. Pathology analysis and immunohistochemistry stains were performed for harvested injured artery segments. The results showed that hVEGF165 could accelerate reendothelialization , attenuate intimal hyperplasia and prevent restenosis.3. The effects of pcDNA3/ hVEGF165 on myocardium angiogenesis and collateral circulation after acute myocardial ischemia. A mouse model of acute myocardial ischemia was made by ligation of left main coronary artery. The pcDNA/ hVEGFlfiS (50ug, N=30) and pcDNA3 (50ug, N=30) were separately injected into the myocardium, where were closed to edge region between infarct zone and non-infarct zone after ligation 20min. The coronary vessel system was imaged by arotic root angiography at the end of 2-week and 4-week. The left ventricular myocardium was harvested for pathologic examination and Northern blot analysis . The angiographic results showed a pronounced accumulation of contrast medium in left ventricular myocardium of experimental group as compared with the control group. The new angiogenesis in the ischemic or infarct myocardium areas were obviously increased in experiment group than that in control group. The results suggusted that pcDNA:,/hVEGF,65 could be transfected intomyocardium and continued to secrete active protein at 4-week and could promote the angiogenesis and collateral circulation in ischemic myocardium.In conclusion, the recombinant eukaryotic expression vector pcDNA3/hVEGF16S can be expressed in eukaryotic cells and...
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